Inhibition, associated with self-efficacy, enables people to control thought and action and inhibit disturbing stimulus and impulsion and has certain evolutionary significance. This study analyzed the neural correlates of inhibition modulated by self-efficacy. Self-efficacy was assessed by using the survey adapted from the Motivated Strategies for Learning Questionnaire. Fifty college students divided into low and high self-efficacy groups participated in the experiments. Their ability to conduct inhibitory control was studied through Go/No-Go tasks. During the tasks, we recorded students’ brain activity, focusing on N2 and P3 components in the event-related potential (ERP). Larger No-Go N2 amplitudes for the high self-efficacy group were found compared with the low self-efficacy group. Conflict detection as represented by N2 was modulated by self-efficacy, whereas conflict inhibition as represented by P3 was not modulated by self-efficacy. The highly self-efficacious students were more capable of detecting conflicts but not necessarily more capable of inhibiting action given that conflict was detected. Taken together, these findings offer neurophysiological evidence of the important regulatory role of self-efficacy in inhibitory control ability development. Inhibition or inhibitory control, referred to as response inhibition, is an executive function defined as the ability to deliberately withhold or override a dominant, prepotent , or automatic response to resist distraction or temptation and to achieve the desired goal . InhibitThe common way to examine the neural correlates of inhibition is using Go/No-Go tasks . In a tyThe ability to conduct inhibitory control is studied through Go/No-Go tasks. The experimental indicators are No-Go N2 and No-Go P3.This study was conducted at a public university of science and engineering in the city of Beijing, China. We used convenience sampling for this study. There were 61 students who had previously been tested for self-efficacy and appropriate samples were selected based on their pretest scores. Twenty-four college students with high self-efficacy were selected, and 26 college students with low self-efficacy were selected. There were 12 females and 12 males, 11 undergraduate students and 13 graduate students in the high self-efficacy group; and 14 females and 12 males, 12 undergraduate students and 14 graduate students in the low self-efficacy group. The experimental participants were right-handed, with normal or corrected vision, and they had no history of mental illness. At the request of the Academic Ethics Committee of the university, they signed informed consent to participate in this study. Self-efficacy was assessed before Go/No-Go tasks by using the survey adapted from the Motivated Strategies for Learning Questionnaire (MSLQ) . The MSLBefore the experiment, the experimental procedure was described and the students participated in the experiment in a relaxed state. Pictures including single triangle and double triangle were selected as the stimulus. The participants were instructed to respond by pressing the button “/” (right hand) or “z” (left hand) as quickly as they could whenever the “Go” stimulus (double triangle) was presented and not to press the button when the “No-Go” stimulus (single triangle) was presented. A fixation cross to orient attention to the middle of the screen was presented for 500 ms. Stimuli were presented for 50 ms, and participants could respond anytime within the onset of the stimulus and the interstimulus interval (950 ms). A practice phase of 20 trials with feedback was given. Each condition consisted of 150 test trials. Go stimuli were presented for most of the trials (80%). All experimental tasks were presented using the E-prime software 3.0.Electroencephalogram (EEG) signals were continuously recorded with the NeuroLab digital amplifier system , using NeuCap with Ag/AgCl electrodes at 32 sites according to the extended international 10–20 system. The reference electrode was placed on the nose tip. The nose-tip reference was converted into bilateral mastoid for reference in offline data analysis. Vertical and horizontal electrooculography (EOG) signals were recorded with two electrodes placed above and below the right eye and with two electrodes at the right and left outer canthi of the eyes, respectively. Data were recorded continuously at a sampling rate of 1,000 Hz and filtered offline with a bandpass of 0.01–100 Hz. Electrode impedance was maintained below 5 kΩ throughout the experiment.1 was used to analyze EEG data. Blinks were corrected using an ICA procedure. The plotted average, condition-specific activation had to account for the N2/P3 time course. The EOG components were identified and selected according to the topographical maps that had to show a fronto-central scalp distribution, which is usually seen with the N2 and P3. One of the EOG components was removed on average. They were performed on all subjects. Remaining artifacts exceeding ± 100μV in amplitude or containing a change of over 100μV within a period of 50 ms were rejected. After performing EOG correction and visual inspection, only artifact-free trials were considered .The EEGLab softwareThe EEG was segmented in epochs of 700 ms, time-locked to picture onset, and included a 100 ms pre-stimulus baseline. Trials contaminated by amplifier clipping, bursts of EMG activity, or peak-to-peak deflection exceeding ± 100 μV were excluded from averaging (excluded 5%). The averaged ERP waveforms were low-pass filtered at 30 Hz. Filter frequencies correspond to the half-amplitude cut-off (24 dB/octave rolloff).For response times, only Go trials with correct responses were included. There were no response times for correct response No-Go trials, as no response was made if participants inhibited the response successfully. Means for all the conditions are presented in post hoc comparisons were performed with Bonferroni correction. Analyses were conducted using the SPSS software .The ERP literature has examined Fz, Cz, and Pz to analyze the inhibition process or self-efficacy effects e.g., . The Fz,F = 4.48, p = 0.04, partial η2 = 0.09) and the main effect of the site  = 6.21, p = 0.003, partial η2 = 0.11), with the Go trials (−2.59 μV) exhibiting smaller amplitude than the No-Go trials (−2.83 μV), and the N2 amplitude at the Cz (- 3.45 μV) significantly larger than those at the Fz (- 1.19 μV) and Pz (- 2.77 μV), there was no interaction among self-efficacy level, Go/No-Go trials, and electrode sites, between Go/No-Go trials and electrode sites, and between self-efficacy and electrode sites. But the main effect of self-efficacy was significant  = 7.12, p = 0.01, partial η2 = 0.13). There is interaction between self-efficacy and Go/No-Go trials  = 4.76, p = 0.03, partial η2 = 0.09). The amplitudes of No-Go N2 were significantly larger in the high self-efficacy group (- 3.42 μV) than in the low self-efficacy group .While repeated-measures ANOVA see  revealedF = 6.74, p = 0.002, partial η2 = 0.12), with the P3 amplitude at the Pz (0.76 μV) significantly larger than those at the Cz (- 0.47 μV) and Fz (- 0.70 μV). A significant interaction effect was found between Go/No-Go trials and electrode sites  = 18.77, p < 0.001, partial η2 = 0.28), with no other interaction effect observed. When we explored each site, self-efficacy level and Go/No-Go trials significantly interacted at only Pz  = 5.02, p = 0.01, partial η2 = 0.12), and an enhanced P3 amplitude on No-Go trials in high self-efficacy group (- 0.30 μV) compared with low self-efficacy group (- 0.61 μV) was identified, but this difference was not significant (p > 0.1).Repeated-measures ANOVA see  revealedThis study investigated the neural correlates of inhibition modulated by self-efficacy. Two groups (low self-efficacy vs. high self-efficacy) of 50 college students participated in the experiments. We adopted Go/No-Go tasks and selected two ERP components associated with inhibitions—N2 and P3. The amplitudes of No-Go N2 were significantly larger in the high self-efficacy group compared with the low self-efficacy group. But there was no significant difference in P3 amplitudes on No-Go trials between the high self-efficacy group and the low self-efficacy group.The finding of larger No-Go N2 amplitudes in the high self-efficacy group indicates that a person with a higher self-efficacy level is more likely to have a stronger ability to detect a conflict. According to In contrast with previous studies, this study found there was no significant difference in No-Go P3 amplitudes between the high self-efficacy group and the low self-efficacy group. The P3 component has been used to ensure that stimulus analyses are appropriately linked with the correct behavioral actions in the monitoring processes; inhibition of the action operation is associated with the No-Go P3 . The finThis study analyzed neural correlates of inhibition modulated by self-efficacy. It was found that students with a higher self-efficacy level tended to have a stronger ability to detect a conflict. Conflict detection was modulated by self-efficacy, whereas conflict inhibition was not modulated by self-efficacy. The high self-efficacious students were more likely to be capable of detecting conflicts but not necessarily followed by inhibition of the action operation.In classrooms, teachers could design meaningful or student-relevant activities to increase their self-efficacy to regulate their behaviors in response to conflicts or challenges, and thus enhance motivational aspects. Teachers also could emphasize the significance of positive reinforcement and a supportive environment, and teach students problem-solving and information processing skills to encourage students to persist longer in learning tasks and engage students actively in class work.This study explored neural correlates of inhibition modulated by self-efficacy based on Go/No-Go task monitoring and processing, but it did not reflect the whole process of inhibition and also focused on a small sample of students. Future research could implement multi-task or more complex measures to assess inhibition modulated by self-efficacy with a larger sample size. We also need to know more about how other predictors contribute further to understanding inhibition behavior change.To sum up, the findings of this study support previous reports that inhibition in response to a stimulus is associated with self-efficacy from the perspective of cognitive neurology, and provide evidence that inhibition, indexed by N2 amplitude, may be one mechanism through which self-efficacy improves task performance. The analysis of ERP components serves to yield a more complete picture of the cognitive mechanism underlying inhibition modulated by self-efficacy. This study provides a new perspective for studies on inhibition and self-efficacy, and it will contribute to our understanding of cognitive ability development.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by China University of Petroleum-Beijing. The patients/participants provided their written informed consent to participate in this study.The author confirms being the sole contributor of this work and has approved it for publication.


The egg production performance of chickens is affected by many factors, including genetics, nutrition and environmental conditions. These factors all play a role in egg production by affecting the development of follicles. MicroRNAs (miRNAs) are important non-coding RNAs that regulate biological processes by targeting genes or other non-coding RNAs after transcription. In the animal reproduction process, miRNA is known to affect the development and atresia of follicles by regulating apoptosis and autophagy of granulosa cells (GCs).In this study, we identified potential miRNAs in the atretic follicles of broody chickens and unatretic follicles of healthy chickens. We identified gga-miR-30a-5p in 50 differentially expressed miRNAs and found that gga-miR-30a-5p played a regulatory role in the development of chicken follicles. The function of miR-30a-5p was explored through the transfection test of miR-30a-5p inhibitor and miR-30a-5p mimics. In the study, we used qPCR, western blot and flow cytometry to detect granulosa cell apoptosis, autophagy and steroid hormone synthesis. Confocal microscopy and transmission electron microscopy are used for the observation of autophagolysosomes. The levels of estradiol (E2), progesterone (P4), malondialdehyde (MDA) and superoxide dismutase (SOD) were detected by ELISA. The results showed that miR-30a-5p showed a negative effect on autophagy and apoptosis of granulosa cells, and also contributed in steroid hormones and reactive oxygen species (ROS) production. In addition, the results obtained from the biosynthesis and dual luciferase experiments showed that Beclin1 was the target gene of miR-30a-5p. The rescue experiment conducted further confirmed that Beclin1 belongs to the miR-30a-5p regulatory pathway.In summary, after deep miRNA sequencing on healthy and atretic follicles, the results indicated that miR-30a-5p inhibits granulosa cell death by inhibiting Beclin1.The online version contains supplementary material available at 10.1186/s40104-022-00697-0. STARts in the granular layer and it is regulated by apoptosis [In modern and intensive animal husbandry, the fertility of animals defines its productivity. Chicken eggs are mostly used for breeding next generations and also serve as protein source for human diets. The growth and development of the ovarian follicle correlates with egg production rate in chickens. The follicles develop continuously and ovulate through periodic selection, whereas the unselected follicles disappear from the ovary by processes involved in follicle atresia. Follicular atresia is a normal process in the ovary to regulate the number of follicles in the developing pool. Mostly, few follicles can successfully develop into pre-ovulation follicle before ovulation. Approximately 99% of the follicles will be subjected to atresia during the follicle developmental process , 2. The poptosis .Follicle development in chickens is a complex biological process regulated by endocrine hormones that are synthesized by the hypothalamus-pituitary-gonad axis (HPG axis) and various paracrine or autocrine factors in the follicles . In addiIn this study, we collected healthy and atretic follicles from laying chickens and broody chickens. The healthy and atretic follicles were ued as two models. Thereafter, we performed miRNA transcriptome sequencing to explore the effects of miRNA on chicken follicle development and atresia. Studies have reported on miRNA transcriptome sequencing in other animal species , 13, howAll animal experiments in this study were performed to meet the requirements of animal welfare. It has been approved by Sichuan Agricultural University Institutional Animal Care and Use Committee with the approval number 2019102013. Tianfu broilers chickens  were the chicken breed used in this study.The follicles used for the deep miRNA sequencing were collected from 200-day-old laying Tianfu broilers and Tianfu Broody Broilers. Three follicular tissues were collected from three chickens per group. The healthy prehierarchical follciles were collected from the laying Tianfu broilers breeders and the atretic prehierarchical follicles were collected from the broody Tianfu broilers breeders. The egg yolk was squeeze off from the granulosa layer and was washed gently with phosphate buffered saline . Thereafter, total RNA was extracted using TRIzol reagent  according to the manufacturer’s instructions. All the RNA samples extracted were stored at − 80 °C for further analysis. The heart, liver, spleen, lung, kidney, breast muscle, leg muscle, brain, fat, intestine, gizzard, ovary, and uterus tissues were also collected from the chickens for further analysis.We construsted cDNA library from the total RNA using Small RNA Sample Pre Kit . Thereafter, preliminary quantification was performed using Qubit2.0  and then the library was diluted to 1 ng/μL. Then, the quality of the library was determined by the size of the library using Agilent 2100 . Afterwards, we performed miRNA and mRNA deep sequencing using the Illumina novaseq6000 (Illumina). Refer to the Gallus_gallus-5.0 (Ensembl release 94) genome for sequencing data analysis. The expression levels of the miRNAs and mRNAs in each tissue sample were statistically analyzed, and the expression levels were normalized using TPM and FPKM algorithm. The TPM normalization formula is as follows:P value), two levels were evaluated and the differential miRNAs were screened. The default differential miRNA screening condition was: P < 0.05. Each comparison combination were given a differential miRNA set, and the union of all the comparison combinations of the differential miRNA sets in each of the experimental group/sample TPM value were used for hierarchical clustering analysis. After obtaining the differentially expressed miRNA between the groups, miRDB, TargetScan and Diana tools were used to predict the target genes of the differential miRNAs.We used DESeq2  based onAccording to the correspondence between the miRNAs and their target genes, we performed Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses on each set of differentially expressed miRNA target genes. The GO enrichment analytical method used was GOseq. In this study, we used KOBAS software to detect the statistical enrichment of the candidate target genes in the KEGG pathway.2 and saturated humidity.The granular layer of the follicles were separated according to Gilbert’s method . The graThe RNA oligonucleotides and overexpression plasmids were designed according to the sequences of miR-30a-5p (miRBase accession number: MIMAT0001135) and Beclin1 (NCBI accession number: NM_001006332.1). Among them, miR-30a-5p inhibitor, inhibitor Negative Control (inhibitor NC), miR-30a-3p mimic, and mimic Negative Control (mimic NC) were constructed using GenePharma . The Beclin1 overexpression plasmid was constructed using Tsingke Biotechnology . Lipofectamine 3000 (Invitrogen) and Opti-MEM (Gibco) were used to transfect the RNA oligonucleotides and overexpression plasmids into the cells.The transfected cells were collected and total RNA was extracted. Then the mRNA was reverse transcripted using Prime Script RT Master Mix Perfect Real Time , whereas miRNA was reverse transcripted using the one-step miRNA cDNA synthesis kit . Primers used for the real-time qPCR were designed using Premier 6 software . The specific information of the primers is shown in Additional file 3: Table SSTAR  was to chemically reacted with horseradish peroxidase (HRP) coupled with the secondary antibody to detect and take photograph of the Western Blot strips. The antibodies used in this study are shown in Additional file 4: Table STotal protein extraction kit  and the BCA protein quantification kit (BestBio) were used for the extraction and quantification of transfected cell proteins. The Western Blot and Immunofluorescence were performed following the methods described previously . In addihttps://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/) was used to verify the target relationship according to the sequences of miR-30a-5p and Beclin1, and then the picture of the target mechanisms was drawn.After detecting that Beclin1 is the target gene for miR-30a-5p, rnahybrid (The Beclin1 3’UTR sequence was inserted into the pcDNA3.1(+) vector by ligase to construct a wild-type or mutant plasmid. Furthermore, the dual luciferase reporter gene was constructed and synthesized using Tsingke Biotechnology (Tsingke), and then it was co-transfected with the RNA oligonucleotides into the granulosa cells. The luciferase activity was detected using Dual-GLO Luciferase Detection System Kit  and the luminescence activity of the firefly luciferase and Renilla was also detected using a multifunctional microplate reader  following the manufacturer’s instructions.We determined the concentrations of the reproductive hormones such as PRL, LH, and FSH in the serum, and the levels of E2, P4, SOD and MDA in the cell supernatant using chicken ELISA kits  following the manufacturer’s instructions.Mcherry-EGFP-LC3 adenovirus was purchased from Hanbio . Specific methods followed were previously described . GCs werThe cell apoptosis was determined using the flow cytometry. 100 μL binding buffer was added to the transfected granulosa cells to resuspend the cells. Then, 5 μL Annexin v-fitc  was added, after staining with fluorescent dye for 10 min in a dark room temperature. Subsequently, 10 μL of PI  was added and stained for 5 min. After avoiding light at room temperature, 400 μL of mixed buffer suspension was added and immediately tested on the machine. Thereafter, CytoFLEX flow cytometer  and Kaluza 2.1 software (Beckman Coulter) were used for detection and data analysis respectively. In addition, ROS was measured using DCFH-DA and the fluorescent probe was also detected using JC-1 to measure the potential of the mitochondrial membrane.t-test was used for comparative analysis between the two groups. One-way ANOVA analysis was used for the comparative analysis of the multiple groups. At P < 0.05, the difference of all statistical tests is considered significant and at P < 0.01 was considered extremely significant. All data are shown as least squares mean ± standard error of mean (SEM).SPSS 19.0 statistical software  was used for the statistical analysis of the data. There were three biological replicates for each experiment. The unpaired Student’s P < 0.01). Furthermore, we found that the levels of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) increased significantly in the laying chicken compared to that of the broody chickens. However, the PRL levels decreased significantly in the laying chicken compared to that of the broody chickens .We compared the phenotypic characteristics and serum hormones of the laying and broody chicken before tissue sampling. We found that there were follicles at different developmental stages in the ovaries of the healthy sexually mature chicken, including pre-hierarchical follicles and hierarchical follicles. However, in the broody chicken, the ovaries were atrophied, and the follicles were all atretic Fig. A and B. https://www.mirbase.org/) [In this study, we performed deep miRNA sequencing analysis on both healthy and atretic follicles obtained from three chickens each per group, thus, normal laying chickens and broody chickens Fig. A. All thse.org/) . We obtaTo study the roles of the miRNAs in the development and atresia of chicken follicles, we selected the 10 highly expressed DEMs as candidates for biological function verification Table A. FurtheThe expression levels of miR-30a-5p in the heart, liver, spleen, lung, kidney, chest muscle, leg muscle, brain, intestine, fat, gizzard, ovary, and uterus were determined, and the results showed that miR-30a-5p was highly expressed in the reproductive organs such as uterus and ovary Fig. A. The prWe predicted that miR-30a-5p might target Beclin1, so we next verified the relationship between miR-30a-5p and Beclin1. To confirm our prediction that miR-30a-5p might target Beclin1, a verification experiment was conducted to determined the relationship between miR-30a-5p and Beclin1. Seed sequence alignment identified miR-30a-5p as a widely conserved miRNA in species including human, mouse, rat, chicken, zebrafish and pig  and autophagy related 7 (ATG7)  Fig. A, whereated Fig. B. Furtheted Fig. C. Furtheted Fig. E. These BCL2. Figure BCL2. The transfected granulosa cells were stained with an annexin V antibody and Propidium Iodide (PI), and then the cell apoptosis was analyzed using the flow cytometry. The results showed that miR-30a-5p inhibitor promoted the apoptosis of granulosa cells, and the apoptotic rate of the granulosa cells decreased after miR-30a-5p mimics transfection , steroid-producing acute regulatory protein (STAR) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) related to the synthesis of E2 and P4 were determined using qPCR analysis. The results as shown in Fig. Granulosa cells are the main cells that synthesize steroid hormones in the follicles. In this study, we determined the effect of miR-30a-5p on the synthesis of estrogen and progesterone in the granulosa cells. The key genes cytochrome P450 family 11 subfamily A member 1  of Beclin1 into the pcDNA3.1(+) vector to construct a Beclin1 overexpression vector. The co-transfection experiments were carried out in three sets. As shown in Fig. 10A and 10B, the results showed that the transfection of Beclin1 overexpression vector promoted the mRNA and protein expression levels of Beclin1, whereas the overexpression of Beclin1 and miR-30a-5p restored the mRNA and protein expression levels. However, the co-transfection experiment showed that the miR-30a-5p inhibited the protein expression levels of the apoptosis marker protein caspase-3 and hinder the conversion of the autophagy marker protein LC3-I to LC3-II . Combined with the KEGG enrichment results of the sequencing data, there are obvious biological differences between healthy follicles and atretic follicles, such as differences in the process of autophagy and steroid hormone synthesis. Therefore, we hypothesized that miR-30a-5p may be a regulator of chicken follicular development and atresia, and then we also found that miR-30a-5p targeted Beclin1. The targeting relationship of miR-30a-5p to Beclin1 was verified in other cells. Studies indicated that miR-30a is down-regulated in liver fibrosis models, and its overexpression prevents liver fibrosis by inhibiting beclin1-mediated autophagy . In card Beclin1 . InteresSTARts at the granular layer and is regulated by apoptosis [BCL2 and downstream caspase activation. This indicated that the accumulation of autophagosomes to a certain level may promote granulosa cells apoptosis [Apoptosis is a basic physiological mechanism to maintain stability in an organism. In mammals, apoptosis was reported to cause follicular atresia . In poulpoptosis . Howeverpoptosis , 32. In There are relatively few studies on the effect of autophagy on broodiness in chickens. However, in geese, hormones and autophagy are important factors that control broodiness. The broodiness in geese is related to an increase in autophagy of granulosa cells and the imbalance of homeostasis of the follicular environment . Other sCYP11A1) catalyzes the first rate-limiting step in the conversion of cholesterol to pregnenolone, whereas cytochrome P450 aromatase (coded by CYP19A1) is involved in the last step of estrogen biosynthesis [Steroid hormones are synthesized from cholesterol through a series of enzymatic reactions. Granulosa cells are mainly responsible for the synthesis of steroid hormones, including estrogen and progesterone. In granulosa cells, a series of enzymes are responsible for converting cholesterol into steroid hormones. Thus, emzymes such as P450scc (encoded by ynthesis , 42. In ynthesis . In concIn this study, we performed deep miRNA sequencing on healthy and atretic follicles. Thereafter, the highly differentially expressed miR-30a-5p from the chicken primary granulosa cells were selected for functional determination. The results showed that miR-30a-5p could inhibit the autophagy and apoptosis of granulosa cells. In addition, miR-30a-5p could promote the synthesis of steroid hormones and increase the level of oxidative stress. The dual luciferase report confirmed that Beclin1 is the target gene of miR-30a-5p. To sum up, miR-30a-5p inhibits granulosa cell death via targeting Beclin1 Fig. . PreviouAdditional file 1: Fig. S1. miRNA expression TPM density distribution map.Additional file 2: Fig. S2. GO analysis of differentially expressed miRNA target genes.Additional file 3: Table S1. Summary of data generated from small RNA deep sequencing.Additional file 4: Table S2. Primers used for quantitative real-time PCR.Additional file 5: Table S3. Antibodies used in article.Additional file 6: Table S4. miR-148a-3p predict target genes.Additional file 7: Table S5. miR-30a-5p predict target genes.


Objective: The frontal QRS-T angle (fQRS-T) is associated with myocardial ischemia and ventricular arrhythmias. On the other hand, acute pulmonary embolism (APE) is a major risk factor for cardiac adverse events. This research aimed to determine whether the fQRS-T, a marker of ventricular heterogeneity, can be used to predict successful thrombolytic therapy in patients with APE.Methods: This was a retrospective observational study. Patients diagnosed with APE and hospitalized in the intensive care unit between 2020 and 2022 were included in the research. A total of 136 individuals with APEs were enrolled in this research. The patients were divided into two groups: thrombolytic-treated (n=64) and non-treated . An ECG was conducted for each patient, and echocardiography was performed.Results: The mean age of the thrombolytic group was 58.2±17.6 years, with 35 females (55.1% of the group) and 29 males (44.9%). The non-thrombolytic group had a mean age of 63.1±16.2, with 41 females (56.5%) and 31 males (43.5%). Respiratory rate, heart rate, and fQRS-T were higher in the thrombolytic group, and oxygen saturation ratio and systolic and diastolic blood pressure were higher in the non-thrombolytic group . In the thrombolytic therapy group, comparing pre- and post-treatment ECG data revealed a statistically significant change in the fQRS-T value (p=0.019).Conclusion: The fQRS-T may provide important clues for the successful treatment of APEs. Acute pulmonary embolism (APE) is a life-threatening cardiovascular disease worldwide . The incDilatation in the ventricles due to afterload causes the remodeling of myocytes . The resIn normotensive patients, anticoagulant therapy prevents thrombus growth and pulmonary embolism (PE) recurrence. In patients with hemodynamic instability, thrombolytic therapy should be administered to reduce PE-related mortality unless contraindicated ,8. SucceThis research aimed to reveal the effect of thrombolytic therapy on the fQRS-T and to determine its association with successful thrombolytic therapy.Study design and subjectThis retrospective investigation was performed in a tertiary hospital. Patients diagnosed with APE and who were admitted to the intensive care unit between January 2020 and June 2022 were included in the research. A total of 136 patients with APE were enrolled in this research. Patients with submassive or massive PE were treated with thrombolytics. The patients were divided into two groups: thrombolytic-treated (n=64) and non-treated patients . Patients with coronary artery disorders, active cancer, hyperthyroidism, mild or severe valve disease, heart failure, chronic renal failure, left ventricular hypertrophy, chronic obstructive pulmonary disease, or congenital heart disease were excluded. Patients whose data were inaccessible and whose analysis was unsuccessful were excluded from this study. The local ethics committee  approved the study protocol (No: 2022-131). It adhered to the Declaration of Helsinki's ethical guidelines for human experimentation (date: 22/07/2022) (2013).Study protocolA 12-lead ECG was obtained using an electrocardiograph  at a rate of 25 mm/s and an amplitude of 10 mm/mV. To digitize the existing ECGs, a scanner was used. At 400x magnification, two cardiologists calculated and analyzed the TpTe time. A significant and practically total agreement was observed between the analyses of the two cardiologists (κ=0.861). The QT interval and TpTe time for heart rate were adjusted using the Bazett formula. During ECG analysis, the QT interval, corrected QT (QTc) interval, QRS, and T axis were automatically identified and recorded. TTE was performed according to the American Society of Echocardiology . The exaDefinitionsThe fQRS-T is the angle formed by the difference between the QRS and T axes Figure .When the angle surpassed 180 degrees, it was changed to the lowest possible angle (360 degrees). The fQRS-T was generated from automatically obtained ECG data.The P dispersion (Pd) was calculated by subtracting the shortest P wave length from the longest P wave duration. The QT and QTc distributions were determined in the same method .Massive PE is defined as acute PE with obstructive shock or systolic blood pressure (SBP) <90 mmHg. Submassive PE is acute PE without systemic hypotension (SBP≥90 mm Hg) but with either right ventricular (RV) dysfunction or myocardial necrosis . StatisticsIBM SPSS software  was used for all the analyses. Initial continuous variables are shown as mean standard deviation or median (interquartile range). Applying Kolmogorov-Smirnov and Shapiro-Wilk tests, the normality distribution of the variables was determined. Categorical variables were represented with frequencies and percentages. Chi-square or Fisher's exact test was utilized for categorical variables. The Student's t-test or Mann-Whitney U-test was used to compare continuous variables. If the p-value was less than 0.05, tests were judged statistically meaningful.The mean age of the thrombolytic group was 58.2±17.6 years, with 35 females (55.1% of the group) and 29 males (44.9%). The non-thrombolytic group had a mean age of 63.1±16.2, with 41 females (56.5%) and 31 males (43.5%). The baseline of the patients is summarized in Table Respiratory rate, heart rate, and fQRS-T were higher in the thrombolytic group; oxygen saturation ratio and SBP and diastolic blood pressure were higher in the non-thrombolytic group . The RV TTE measurements demonstrated worsened RV functions in the thrombolytic group . Patients' electrocardiographic parameters were analyzed and evaluated Table .In the thrombolytic therapy group, comparing pre- and post-treatment ECG data revealed a statistically significant change in the fQRS-T value (p=0.019).This research revealed that the fQRS-T, which increased as a result of ventricular loading due to APE, decreased both clinically and statistically after successful thrombolytic therapy.Thromboembolism causes an increasing afterload after dilatation in the right ventricle. The echocardiographic findings in our study support right ventricle dilatation in the thrombolytic group. Higher oxygen consumption is caused by shear stress in the ventricular wall, neurohormonal activation, and tachycardia . HypoxemIn clinical practice, discovering novel ECG characteristics, such as fQRS-T, may be valuable for defining massive and submassive PE and predicting the effect and success of thrombolytic treatment in patients with APE. The fQRS-T, an indication of heterogeneity in ventricular activation and conduction, is the angle between ventricular depolarization and repolarization . There aIn a study, fQRS-T was shown to be a predictor for mortality in patients with acute coronary syndrome . Gül et LimitationsThe population of the study was small. In this study, the estimated fQRS-T values for both groups were observed to be lower than those previously reported in studies on cardiovascular disorders. This difference is due to the exclusion of participants with severe cardiovascular disease. The absence of a significant change in the frontal QRS-T angle in the non-thrombolytic patient group in the intensive care unit may be explained by the negligible effects of PE on the right ventricle or by the fact that the therapy had no impact on the right ventricle.This research indicated that the fQRS-T is an important indicator of ventricular loading in patients with APE. However, the non-invasive and comprehensible fQRS-T method in clinical follow-ups may provide important clues for the successful treatment of APE. Adverse events could be prevented by using this novel marker.


Drug-coated balloon (DCB) has been proved efficacy for coronary small vessel disease, but data regarding outcomes of DCB in common de novo lesions (including reference vessel diameter more than 3.0mm) compared with new-generation drug-eluting stent (DES) are lacking. We hypothesized that a DCB-only strategy for coronary de novo lesions would be non-inferior to DES treatment on angiographic outcomes.In this randomized controlled trial, we compared the effect of DCB with DES on late lumen loss (LLL) at 9-month angiographic follow-up and 12-month major adverse cardiac events (MACEs), including death, non-fatal myocardial infarction, target lesion revascularization (TLR), and target vessel revascularization (TVR).P=0.019). The 12-month cumulative MACE rate was similar in the DCB and DES groups .From July 2017 to July 2018, 288 consecutive patients with reference vessel diameter (RVD) between 2.25 and 4.0mm were screened. After proper pre-dilation, 170 patients were enrolled and randomized to the DCB and the DES groups at 1:1 ratio. Seven patients withdrew the consent forms during hospital stay . Two patients in DCB group underwent bailout stenting due to severe dissection after DCB release. The primary endpoint of 9-month LLL was −0.19±0.49mm with the DCB versus 0.03±0.64mm with the DES. The one-sided 97.5% upper confidence limit of the difference was −0.04mm, achieving non-inferiority of the DCB compared with the DES (In this prospective study, the DCB only strategy for de novo lesion was non-inferior to the new-generation DES in terms of 9-month late lumen loss.The online version contains supplementary material available at 10.1007/s10557-021-07172-4. Drug-coated balloon (DCB) is recommended treatment for in-stent restenosis  and alsoThis prospective, randomized, open-label, single-center study was designed to evaluate the efficacy and safety of DCB in the treatment of coronary de novo lesions. Patients who underwent elective percutaneous coronary intervention (PCI) at Beijing Hospital were screened. The target lesions should not be intervened before, with a RVD between 2.25 and 4.0 mm and lesion length of ≤30 mm. The patients were consecutively enrolled and randomized into DCB and DES groups at a 1:1 ratio if the pre-dilation achieved ideal results  of target lesions at the 9-month angiographic follow-up. LLL was defined as the minimal lumen diameter (MLD) immediately after the procedure minus the MLD at 9 months. The secondary endpoint was the major adverse cardiovascular events (MACE) after 12 months. MACE was defined as the composite of cardiac death, non-fatal myocardial infarction, target vessel revascularization (TLR), and target vessel revascularization (TVR). Cardiac death was defined as any death that was not clearly of extracardiac origin, and myocardial infarction, according to guidelines. Myocardial infarction was defined as evidence of myocardial necrosis consistent with the third International Definition of Myocardial Infarction . TVR wasPCI was done under DAPT with aspirin (100 mg/day) and clopidogrel (75 mg/day) or ticagrelor (90 mg twice a day). Before PCI, 70–100 U/kg of unfractionated heparin was administered, with an additional 1000 U for every extra hour of procedural time. Glycoprotein IIb/IIIa antagonists were administered at the operator’s discretion. Non-target vessel lesions were to be treated before the target vessel lesion intervention with no complications. Otherwise, the patients were excluded. Patients in the DCB group with stable coronary disease received DAPT for 1–3 months  or for 6Appropriate pre-dilation was performed before using a paclitaxel-coated balloon catheter with a balloon/vessel diameter ratio of 0.8–1.0, according to the recommendations of the German  and ChinA total of 170 eligible patients enrolled were randomized to receive the DCB or DES in a 1:1 ratio using a random number. Number randomization was performed by a statistician using SPSS 22.0 software . This trial was open labeled; therefore, participants or investigators were not masked to the treatment.All angiograms were carefully recorded in all critical periods. At least 2 orthographic views (reference views) were required in pre-procedural angiograms at lesion site, accurate DCB location angiograms obtained before dilation, and 2 post-procedural angiograms with a similar projection angle as the pre-procedural angiograms. Follow-up angiograms were recorded with a similar projection angle as the post-procedural angiograms. All lesions were analyzed using the software built in Allura Xper FD20 flat-panel digital cardiac system . Two independent technicians who were not involved in the intervention measured and recorded the RVD, MLD, lesion length, percent diameter stenosis (DS%), and percent area stenosis. Measurements were performed in triplicate and the mean value was recorded.t-test with a one-sided P value <0.025. Non-inferiority for in-device late loss was declared if the upper limit of the one-sided 95% confidence interval (CI) difference in late loss (DCB minus DES) did not exceed a delta of 0.15 mm from the observed late loss in the DES group according to results reported previously  despite the similar pre-procedural RVD. A certain amount of residual stenosis was allowed after DCB treatment   mm] was significantly lower than that of the DES [3.00 (2.75 to 3.50) mm] . The procedural success rate in the DCB group was 97.6% (82/84), similar to that in the DES group 98.7% (78/79).P <0.001).There was no difference in pre-intervention MLD between the two groups, but the post-intervention MLD (1.82±0.43mm vs. 2.54±0.50mm) and the immediate lumen gain (0.84±0.56mm vs. 1.53±0.64mm) were significantly lower in the DCB group than those in the DES group (both P <0.001), while this trend was not observed in the DES group . The primary endpoint of 9-month LLL was −0.19±0.49mm with the DCB versus 0.03±0.64mm with the DES. The 95% CI of the difference was −0.40mm to −0.04mm, achieving non-inferiority of the DCB compared with the DES (P=0.019). Comparing with those in the DES group, the follow-up MLD was significantly lower and the DS% was more severe in the DCB group, which was considered due to the lack of supporting structure after DCB delivery. However, the restenosis rate (defined as DS% ≥50%) was similar between the DCB and the DES groups   completed angiographic follow-up at an average of 9 months. Two patients were dropped-out due to DCB failure and 9 patients refused angiographic follow-up due to lack of symptoms, financial difficulties, or fear of invasive procedures. The angiographic success rate was 94.4% (152/161). The 9-month follow-up MLD in the DCB group was significantly increased compared with post-intervention level   and 6.33% (5/79) respectively, showing no significant difference . When discussing the surrogate endpoints for DES clinical trials, Pocock et al. pointed out that the TLR rate would be very low if DS% in the stented segment was <30% at follow-up, and a lower DS% would not further reduce adverse clinical events [The recently published RESTORE SVD China study used DS% as the primary end point . The autl events . WhetherThere was no difference in the 12-month clinical endpoint between the two groups in this study, which was consistent with the non-inferior DCB angiographic results. Similar results have also been reported in recent observational studies. In the DEBATE study , 120 patP=0.025) respectively, suggesting that the angiographic success criteria for the two procedures are different. The main function of DCB is to deliver the drug to the lesion and transfer it into the vessel wall [The key to successful DCB treatment for coronary de novo lesions is achieving desirable pre-dilation. Most consensus statements , 16, 26 sel wall , rather Hermans et al. showed that after successful POBA , the restenosis rate was 29% in patients with dissection and 30% in those without dissection, and dissection did not increase the incidence of MACE , 9. ThisThere were several limitations in this study. First, the study was powered for the primary endpoint but was not insufficient to verify the differences in clinical endpoint events. Second, single-center study would introduce bias. Third, the DAPT duration in the DCB group showed great variability, with patients receiving treatment for 3 months if they had no previous PCIs or PCI more than 12 months before enrollment, while others received DAPT for 3–12 months if they had undergone DES implantation within 12 months of enrollment. This heterogeneity may affect the clinical endpoint. Fourth, we did not restrict the type or number of balloons used for pre-dilation and post-dilation since physicians have their own preferences. We also did not specify the stent brand, considering that the efficacy of the new-generation DES used at our center seemed equivalent; however, differences may exist.This prospective single-center randomized study showed that paclitaxel DCB only strategy for coronary de novo lesions is noninferior to the new-generation DES in term of LLL at 9 months. Moreover, there was no significant difference in MACEs rate at 12 months.ESM 1(DOCX 27 kb)


This confirms that the developed hybrid membranes can be efficiently used to separate water from azeotropic aqueous ethanol.Chitosan-capped silver nanoparticle (CS-capped AgNPs)-incorporated Poly (PVA) hybrid membranes were prepared by a solution-casting technique for ethanol dehydration via pervaporation. The incorporation of CS-capped AgNPs into the PVA membrane and its influence on membrane properties and pervaporation-separation process of azeotropic water/ethanol mixture was studied. The addition of CS-capped AgNPs into the PVA membrane reduced the crystallinity, thereby increasing the hydrophilicity and swelling degree of the hybrid membrane, supported by contact angle (CA) analyzer and swelling degree experiments, respectively. Fourier transform infrared spectroscopy (FTIR) demonstrated the formation of polymeric matrix between PVA and CS and also the binding of AgNPs onto the functional group of CS and PVA, which was also reflected in the microstructure images demonstrated by scanning electron microscopy (SEM) and by 2θ angle of wide-angle X-ray diffraction (WAXD). The effect of CS-capped AgNPs on the thermal stability of the hybrid membrane was demonstrated by differential scanning calorimetry (DSC) and thermogravimetric analyzer (TGA). These characteristics of the hybrid membrane positively impact the efficiency of the dehydration of ethanol, as indicated by pervaporation experiments. The best performances in total flux (12.40 ± 0.20 × 10 Ethanconcerns ,11. Instconcerns .Pervaporation (PV), in its simplest type, is a combination of membrane permeation and evaporation associated with lower energy consumption and an economical process ,14,15. PIn the PV process, Poly (PVA) is a commonly used polymer for developing membranes. Advantages of using PVA membranes in the PV process are their non-toxic nature, water solubility due to the presence of the hydroxyl group and being biocompatible and biodegradable synthetic polymers ,22,23. TOn the contrary, hybrid materials combine the important properties of both inorganic and organic and create new compositions with unique characteristics, which offer specific advantages for fabricating PV membranes exhibiting high selectivity and flux with desirable thermal and chemical strength. Perhaps, this becomes a facile and feasible way to solve the trade-off phenomenon, which generally exists in the PV process ,30,31.−2 h−1. Jiang et al. [−2 h−1) and selectivity (92). Xia et al. [−2 h−1. Our previous work [2SA-grafted Poly–graphene hybrid membranes for ethanol dehydration by PV. Among these, the membrane containing 2 wt.% of graphene exhibited selectivity of 4187 with a flux of 0.1166 kg.m−2 h−1.Considering this, Wang et al.  developeg et al.  had overa et al.  developeous work  fabricatChitosan is the most abundant biopolymer and is deacetylated form of chitin. It exhibits hydrophilicity, degradation and antibacterial properties. It can adsorb metal ions using chelation due to the lone pair of electrons in its amino nitrogen ,39,40,41Silver nanoparticles (AgNPs) are used in different fields, including medical, food, health care, consumer and industrial fields, due to their unique physical and chemical properties. These include optical, electrical and thermal properties, high electrical conductivity, biological properties ,43,44 anIn the present investigation, by understanding the pros and cons of developing hybrid membranes, we attempted to develop hybrid membranes by suitably incorporating CS-capped AgNPs. Different characterization methods are used to examine the physical and chemical properties of the fabricated membranes. Effects of CS-capped AgNPs and temperature on PV performance were systematically studied. The performance of fabricated membranes was evaluated by correlating with their structural properties.−1 in the FTIR spectra of the PVA membrane, and multiple brands appeared between 1000 to 1200 cm−1 corresponding to –C-O stretching vibrations of the hydroxyl groups. These bands matched those reported by Sajjan et al. [−1, and bands appeared at around 1645 and 1550 cm−1 corresponding to NH (amide I) and amide II functional groups of the pure chitosan [n et al. . The widchitosan ,48.−1, showing the movement towards the higher wavenumber along with the increase in the intensity of the band as we progressed from M-1 (0.5% CS-capped AgNPs) to M-4 (2 wt.% CS-capped AgNPs), indicating enhanced hydrogen bonding due to PVA-CS chain interactions [−1, a new peak appeared. Its peak intensity increased as the wt.% of CS-capped AgNPs increased in the PVA matrix, indicating that silver particles are bound to the functional groups present in chitosan and PVA. The shifting of the peak is due to the formation of a coordination bond between the silver atom and the electron-rich group present in PVA and chitosan [In the case of CS-capped AgNPs incorporated hybrid PVA membranes, broadband appeared at 3433 cmractions . Multiplchitosan ,49θ = 20°, which indicates the presence of crystalline and amorphous regions in the morphology of the membrane [The illustrations of the WAXD pattern of PVA and CS-capped AgNPs embedded in PVA membranes are illustrated in membrane . The difmembrane . The peaFurther, incorporated CS-capped AgNPs disturbed the PVA chains due to the intermolecular hydrogen bonding between PVA and CS-capped AgNPs and electrostatic interaction with AgNPs, responsible for decreased crystallinity of the PVA membrane; hence, CS-capped AgNPs membranes have higher water-retention capacity. The second stage of decomposition of the PVA membrane starts from 283–465 °C, attributed to major weight loss due to the decomposition of polymer chains . Compareg) investigation was performed to characterize the thermal behavior of the produced membranes. The membrane thermograms  to M-4 (2 wt.% CS-capped AgNPs). The interaction between PVA and CS-capped AgNPs causes chain mobility to be hampered, resulting in an increase in Tg. When the proportion of CS-capped AgNPs in the PVA membrane matrix increased, the Tg values increased from M-1 (0.5 wt.% CS-capped AgNPs) to M-4 (2 wt.% CS-capped AgNPs), indicating that the PVA membranes became more stable.The glass-transition-temperature , temperature (p = 1.76 × 10−14 & 2.46 × 10−48 < 0.05 = α), an interaction effect between membrane type and temperature (p = 0.0432 & 6.46 × 10−40 < 0.05 = α) shows that the factors are statistically significant by rejecting the null hypothesis. To see the difference in means of the parameters between membrane type and temperature, a post hoc test using Tukey’s HSD was performed after the two-way ANOVA. A Tukey test on membrane type showed a significant effect on total permeation flux and selectivity, as means between all membranes were higher than the mean critical of 0.002576 for total permeation flux and 9.3161 for selectivity, respectively. Similarly, temperature showed a significant effect on total permeation flux and selectivity, as means between all temperatures were higher than the mean critical of 0.001696 for total permeation flux and 6.132 for selectivity, respectively. However, the Tukey test showed no significant effect between membrane type and temperature for total permeation flux and selectivity.Two-way ANOVA with replication was performed at a 95% (α = 0.05) confidence level to see the effect of membrane type and temperature on permeation flux and selectivity, as represented in The pervaporation-separation index (PSI) is the product of total permeation flux and selectivity, which characterizes the membrane-separation ability. This index can be used as a relative guideline for designing new membranes for the pervaporation-separation process and selecting a membrane with an optimal combination of flux and selectivity. To assess the overall performance, the calculated PSI data are plotted as a function of wt.% of CS-capped AgNPs for the azeotropic water-ethanol mixture . It is o2 groups on the membrane. These are responsible for predominating the plasticizing effect on the membrane due to greater swelling. Therefore, the permeation of diffusing molecules and the associated molecules through the membrane becomes easier, increasing total permeation flux while suppressing the selectivity.The effect of operating temperature on the PV performance for the azeotropic water-ethanol mixture was studied for all the membranes, and the resulting values are presented in −2 kg/m2 h) and selectivity (3612.33 ± 6.03) at 30 °C for 10 mass% of water in the feed. Experimental data also revealed that total flux and water flux are almost overlapping for all the CS-capped AgNP-incorporated membranes, suggesting that the developed membranes have higher separation ability. The temperature effect study indicated that permeation flux was increased while decreasing selectivity when the operating temperature was increased. This is mainly because of decreased viscosity of permeating molecules.In this study, new hybrid PVA membranes were prepared by incorporating different wt.% of CS-capped AgNPs for applications in ethanol dehydration from azeotropic water/ethanol mixtures. Increased CS-capped AgNPs content in the membrane matrix resulted in simultaneous permeation flux and selectivity increases. This was explained based on the significant enhancement of hydrophilicity and the amorphous nature of the membrane matrix. The PV separation-index data also indicates that the membrane incorporated with 2 wt.% CS-capped AgNPs (M-4) showed an excellent performance while separating the water-ethanol mixtures because of enhanced hydrophilicity and selective interaction between the membranes and permeate. The membrane M-4 showed the highest total flux , PVA , and Acetic acid (purity ≥ 99.5%) were purchased from s.d.fine Chemicals Ltd., Mumbai, India. Chitosan, CS  and silver nanopowder  were purchased from Sigma-Aldrich Chemicals, St. Louis, MO, USA. Rectified ethanol (95.6 vol.%) from Godavari Biorefineries, Karnataka, India. Chemicals purchased were used without further purifications as they are reagent-grade chemicals.The purpose of fabricating CS-capped Ag nanoparticles was to improve the PVA membrane in terms of PV performance. The fabrication procedure was as follows: In 50 mL of water, 2 vol.% acetic acid and 1 g chitosan were added and mixed for 24 h at room temperature. A total of 0.25 g of AgNPs was combined in this homogeneous solution and agitated continuously for 24 h at 60 °C to generate a suspension of CS-capped AgNPs. This dispersion was then sonicated for 30 min at a constant frequency of 38 kHz in an ultrasonic bath  to break up any possible silver-nanoparticle clumps.Poly (4 g) was dissolved in deaerated water (96 mL) and agitated at 30 °C for 24 h. Further, the solution was filtered, and the undissolved particles were removed. The solution was then gently spread out on a glass plate and allowed to dry for 72 h. The membrane was then carefully peeled away and labeled M.The development of the CS-capped AgNPs-Poly membrane was carried out according to the procedure mentioned ahead: To uniform PVA solution, a fixed amount of CS-capped AgNPs suspension was mixed and stirred for 24 h. The prepared mixture was then passed through a sonication process maintained at 40 kHz for 2 h for proper distribution of CS-capped AgNPs in the base matrix (PVA). The prepared solution was cast on a clean glass plate. The rest of the procedure is similar to the procedure followed for the fabrication of M. Known amounts  of CS-capped AgNPs were added, and the obtained membranes were named M-1, M-2, M-3 and M-4. The breadth of the fabricated membranes was measured with a thickness gauge at several locations with a precision of 2 µm. . A constant thickness of 50 ± 2 µm was achieved. The scheme of the fabrication of CS-capped AgNP-incorporated PVA membrane is shown in −1 and with a spectral resolution of 4 cm−1.The structural interactions between CS-capped AgNPs and PVA membrane were studied by Spectrum two with Diamond ATR Fourier transform infrared (FTIR) spectroscopy . FTIR analysis was recorded in the range of 500 to 4000 cmA wide-angle X-ray diffractometer  was used to identify the solid-state morphology of the membranes developed. Ni-filtered CuKα radiation (30 mA) was used as an X-ray source, and a cathode current of 40 kV was applied. Samples of membranes in the range of 5° to 70° were scanned at the rate of 8°/min for the angle 2θ. Differential scanning calorimetry (DSC) measurements  and Thermogravimetry analyzer (TGA)  was used for thermal analysis of the fabricated membranes. Scanning electron microscope (SEM)  was used to study the surface morphology of the fabricated membranes.In order to study the water affinity of the fabricated membranes at 30 °C, the contact angle was measured, employing a contact-angle meter named Acam-series, Apex Instruments Co. Pvt. Ltd., Kolkata, India. In order to further confirm the hydrophilic nature of the developed membranes, membrane-sorption analysis was conducted according to the procedure mentioned in the previous study .2. A circulation pump with adjustable speed was equipped on the feed side. A vacuum of 31.325 kPa was kept on the permeate side using a vacuum pump equipped with a pressure gauge (PN2299 pressor sensor with display). The pressure gauge was also equipped with a pressure sensor to maintain the system pressure constant. The stirrer was fixed in the feed container to keep the same temperature throughout the tank.For PV experiments, a specifically designed PV apparatus was used, and the photographs of the PV unit are shown in After the membrane reached equilibrium, a fixed vacuum was induced, and on the permeate side, the vapors were accumulated after condensation in the cold traps by inserting liquid nitrogen at uniform intervals. Subsequently, permeate weight was measured by using a digital microbalance, and the permeation flux was calculated. KAFI smart Karl Fischer Titrator measured the composition of the permeate. A minimum of 3 different readings were taken, and the mean value was considered for parameters such as permeation flux, selectivity and PSI. The PV performance of the fabricated hybrid membranes was analyzed by calculating the total permeation flux, separation selectivity and pervaporation-separation index (PSI) using the equations reported in the literature ,55,56,57


Cuproptosis is a newly discovered new mechanism of programmed cell death, and its unique pathway to regulate cell death is thought to have a unique role in understanding cancer progression and guiding cancer therapy. However, this regulation has not been studied in low grade glioma (LGG) at present. In this study, data on low grade glioma patients were downloaded from the TCGA database. We screened the genes related to cuproptosis from the published papers and confirmed the lncRNAs related to them. We applied univariate/multivariate, and LASSO regression algorithms, finally identified 11 lncRNAs for constructing prognosis prediction models, and constructed a risk scoring model. The reliability and validity test of the model indicated that the model could well distinguish the prognosis and survival of LGG patients. Furthermore, the analyses of immunotherapy, immune microenvironment, as well as functional enrichment were also performed. Finally, we verified the expression of these six prognostic key lncRNAs using real-time polymerase chain reaction (RT-PCR). In conclusion, this study is the first analysis based on cuproptosis-related lncRNAs in LGG and aims to open up new directions for LGG therapy. Gliomas are the most common and long-lasting malignant primary brain tumors , includiIn 2012, Columbia University discovered a new type of programmed cell death, ferroptosis, which is completely different from apoptosis, programmed death, and pyroptosis in its mode of action and molecular mechanism. A type of programmed cell death induced by excessive accumulation of lipid peroxides . In 2019Long non-coding RNAs (lncRNAs) are a class of RNAs longer than 200 nucleotides without the capacity to encode for protein. They have important functions in transcriptional silencing, transcriptional activation, chromosome modification, and intranuclear transport and perform important regulatory capabilities . A largeAs the first biological study to integrate lncRNAs with cuproptosis in LGG, we aim to establish a new signature of cuproptosis-related lncRNAs to predict the prognosis of LGG patients and to evaluate the efficacy of these lncRNAs. Based on the TCGA database, we finally screened 11 of them and constructed a risk model to predict OS in LGG patients. We then divided LGG patients into two groups (high-risk and low-risk) based on mean-risk scores. Through the analysis, we found that high-risk groups had better responses to immunotherapy, suggesting that the model could serve as a potential biomarker to predict the prognosis of LGG patients. We hope that our study will improve the accuracy of prognosis prediction and elucidate the possible mechanism of cuproptosis-related lncRNAs in LGG.https://cancergenome.nih.gov/). Ten cuproptosis-associated genes including FDX1, LIAS, LIPT1, DLD, DLAT, PDHA1, PDHB, MTF1, GLS, and CDKN2A were acquired from the literature  and the testing set (n = 239). The baseline characteristics of the training set and the testing set are shown in p > 0.05). The prognostic values of each lncRNA were first evaluated using univariate Cox regression analysis. The least absolute shrinkage and selection operator (LASSO) Cox regression were performed to reduce the dimension of high-latitude data using R package “glmnet”. Ten-fold cross-validation was employed to avoid the overfitting problem and select the penalty parameter (λ) according to the minimum criteria. Multivariate Cox regression analysis was then performed to identify the final candidates involved in the risk signature. The testing set and the entire set were used to validate its accuracy of it. The risk score for each LGG patient was counted with the following algorithm: risk score = expression of a lncRNA [1] × corresponding coefficient [1] + expression of a lncRNA [2] × corresponding coefficient [2] + expression of a lncRNA [n] × corresponding coefficient [n]. Additionally, we classified the LGG patients into two groups based on their risk scores: low-risk group and high-risk group.We randomly divided the 479 LGG samples into the training set (t-distributed stochastic neighbor embedding (t-SNE)   and t-di (t-SNE)  analysisTo determine whether the risk score was superior to other clinical traits, an independent prognostic factor analysis utilizing univariate and multivariate Cox regression was conducted in R with the package “survival”. Using R package “RMS”, we established a nomogram integrated risk scores as well as other clinicopathological characteristics  to better predict the 1-, 3-, and 5-year OS. Besides, we applied calibration curve analysis to examine the reliability of the established nomogram. The prediction accuracy was examined by using the receiver operating characteristic (ROC) curves and conformance index (C-index).We applied the estimate algorithm to calculate each patient’s immune score, stromal score, and estimate score using the R package “ESTIMATE” . FurtherWe applied VarScan software to process the “mask somatic mutation” data from the TCGA database . The tumThe IC50 was calculated using the R package “pRRophetic”, and the chemotherapeutic medications were obtained from the Genomics of Drug Sensitivity in Cancer (GDSC) database . The IC5−ΔΔCt method with each test performed in triplicate. The primer sequence is shown in All tissue samples were collected from the Neurosurgery Department of Qingdao University Affiliated Hospital, which was approved by the Medical Ethics Committee of the hospital. All specimens were stored in liquid nitrogen, and all patients provided informed consent. From July 2022 to August 2022, four low grade glioma tissue samples and four non-tumor brain tissues were obtained. Total RNA was isolated using TRIzol reagent, quantitative real-time fluorescence quantitative PCR(qRT-PCR) was performed by using primers (Beijing Genomics institution) and premix (SYBR Master Mix). Data were analyzed using the 2p-value < 0.05). GO and KEGG enrichment analyses were applied using the package “clusterProfiler” in R. We performed a GSEA analysis using the GSEA software  to further screen the difference of functional pathways between high-risk and low-risk groups. The network among lncRNAs, mRNAs, and risk types was visualized by Cytoscape. Furthermore, the correlation between cuproptosis-related lncRNAs, cuproptosis associated genes, and factors with risk or protection was analyzed using the R package “ggalluvial”.We applied the “limma” R package to distinguish the differentially expressed genes (DEGs) in the subgroups following criteria (|log2-fold change (FC)| ≥ 1, Pearson R| > 0.4 and p < 0.001) . The Sankey diagram in The workflow of this study is shown in < 0.001) . The SaTo identify the ideal risk signature to guide the prognosis in patients with LGG, the univariate Cox regression analysis was conducted to screen for 297 cuproptosis-related lncRNAs associated with OS in concert with training set information . FurtherThe risk score was produced with the formula: risk score = expression of MUC12-AS1 × 0.382030727494924 + expression of AL158212.3 × -1.39920798375446 + expression of BASP1-AS1 × -1.9645994664291 + expression of AL589843.1 × 0.758072939276948 + expression of AC099850.3 × 0.311101322216435 + expression of AC017104.1 × 0.654061856724554 + expression of AL162511.1 × -0.688335466581498 + expression of AC244453.3 × 2.05053269718406 + expression of FAM181A-AS1 × 0.515445156843301 + expression of AC002351.1 × 0.556137908535174 + expression of LINC01537 × -1.84136636638082.p < 0.001) in OS between high- and low-risk groups in the K-M analysis curve  and the entire set . Additionally, the AUCs for 1-, 3-, 5-year OS rates were 0.818, 0.754, 0.679, respectively based on the testing set . Likewise, in the multivariate Cox analysis (p < 0.001). As nomograms is widely used to predict the survival and prognosis of cancer patients. Then we established a nomogram using the risk score and clinicopathological features such as gender, grade, and age to better estimate the 1-, three- and 5-year survival rate for LGG patients . The survival of LGG patients in the high TMB group is better than that of the patients in the low TMB group. Additionally, the patients in the high-risk group with the high TMB group show a worse survival time, and the patients in the low-risk group with the low TMB group hold a significantly better OS , Dabrafenib, and lncRNA LINC01537 was the second , and so on. We speculated that the immunotherapy responses might be different between the low-risk and high-risk groups because of the significant differences in the immune microenvironment. To explore potential drugs targeting our risk model and improve treatments for patients with LGG, we used the IC50 values obtained from 138 patients using the “pRRophetic” package to determine therapeutic response. The IC50 of A.770041, AKT. inhibitor.VIII, AP.24534, AS601245, ATRA, AUY922, AZ628, and AZD.0530 were significantly higher in the low-risk group, suggesting that LGG patients in the high-risk group might benefit from those possible anti-tumor drugs (p < 0.05). Thus, we may be able to select the most appropriate drugs for LGG patients, as well as our risk model based on the cuproptosis-related lncRNAs, which may provide a powerful cue for immunity therapy guidance.We performed the drug sensitivity analysis to explore the high correlation drugs targeting 11 cuproptosis-related lncRNAs . The resWe selected six Cuproptosis-related lncRNAs for validation, and we detected their expression levels in non-tumor brain tissues and glioma tissues, By using RT-qPCR assay, the expression level of AL158212.3,BASP1-AS1, LINC01537 showed an overall upward trend in non-tumor brain tissues, the expression level of AC099850.3,AC244453.3,FAM181A-AS1 showed an overall upward trend in glioma tissues. However, through further verification, there is no significant difference in the expression level of AC244453.3 between non-tumor tissues and LGG, which may be due to the small number of samples .LGG is heterogeneous and highly aggressive, and patient treatment and prognosis prediction are the focus . HoweverIn recent years, more and more researchers have discovered that long non-coding RNAs (LncRNAs) play an important role in the progression and metastasis of malignant tumors . MoreoveIn our study, we identified 1149 cuproptosis-related lncRNAs, of which 11 cuproptosis-related lncRNAs were screened as OS prediction models in LGG patients. Risk signature screening was performed for seven risk factors (MUC12-AS1 AL589843.1 AC099850.3 AC017104.1 AC244453.3 FAM181A-AS1 AC002351.1) and four protective factors (AL158212.3 BASP1-AS1 AL162511.1 LINC01537). Some of these lncRNAs appeared in the construction of prognostic prediction models. For example, BASP1-AS1 is positively correlated with the prognosis of LGG and may be a target for LGG therapy . Jiang aWhen we screened drug candidates by assessing the response of the samples against the GDSC database, we were pleasantly surprised to find that the low-risk group was more resistant to all compounds, implying that high-risk patients may have a higher susceptibility to these drugs. Immunotherapy is promising in the treatment of brain tumors, and predictive biomarkers have been shown to play a role in the immunotherapy of tumor patients. For example, the oncogenic activation pathway PI3K-AKT-mTOR pathway was shown to induce ferroptosis in LGG cells; This can help predict disease outcome and response to treatment . In a st+ T cells, and macrophages in LGG (The number of coding-competent bases subject to somatic mutation is called TMB. TMB can induce the formation of new antigens, thereby triggering antitumor immunity. TMB can serve as an effective biomarker for predicting response to immunotherapy . In our s in LGG . As mentFrom a clinical perspective, the pathological stage can be a decisive factor in the prognosis of LGG patients. However, due to the present periodization, systems are incompetent to accurately predict the prognosis of LGG patients. LGG patients always have different clinical outcomes irrespective of the same stage. So, novel predictive biomarkers are urgently needed. Here, we established a cuproptosis-related lncRNAs model that can predict the prognosis of LGG patients. Our study also offers insights for further understanding the mechanism of cuproptosis. We utilized multiple methods to support the reliability of the model and optimized it. We assumed that the model was dependable without external data validation. We do aware that there are some limitations in this study. First, additional clinical datasets are required to validate the model. Second, the molecular biological function of cuproptosis-related lncRNAs has not been fully explored. Thus, we’d like to recollect clinical samples to further confirm our study. Besides, we will perform a further study to explore the molecular biology functional mechanism of cuproptosis-related lncRNAs in LGG.In conclusion, we constructed a prognostic model that is sensitive and reliable, and it showed well efficacy in predicting the prognosis of patients with LGG. Additionally, the model can also predict the response to immunotherapies of LGG patients. Subsequently, we screen potential drugs to improve current treatment management. On the other hand, our study laid the groundwork for exploring the role of cuproptosis in the biogenesis and progression of LGG.


Extracorporeal shockwave therapy (ESWT) can stimulate processes to promote regeneration, including cell proliferation and modulation of inflammation. Specific miRNA expression panels have been established to define correlations with regulatory targets within these pathways. This study aims to investigate the influence of low-energy ESWT—applied within the subacute and chronic phase of SCI  on recovery in a rat spinal cord contusion model. Outcomes were evaluated by gait analysis, µCT and histological analysis of spinal cords. A panel of serum-derived miRNAs after SCI and after ESWT was investigated to identify injury-, regeneration- and treatment-associated expression patterns. Rats receiving ESWT showed significant improvement in motor function in both a subacute and a chronic experimental setting. This effect was not reflected in changes in morphology, µCT-parameters or histological markers after ESWT. Expression analysis of various miRNAs, however, revealed changes after SCI and ESWT, with increased miR-375, indicating a neuroprotective effect, and decreased miR-382-5p potentially improving neuroplasticity via its regulatory involvement with BDNF. We were able to demonstrate a functional improvement of ESWT-treated animals after SCI in a subacute and chronic setting. Furthermore, the identification of miR-375 and miR-382-5p could potentially provide new targets for therapeutic intervention in future studies. Extracorporeal shockwave therapy (ESWT) has proven to be effective for various pathologies in different tissues ,3,4,5,6.Shockwaves are defined as types of acoustic pressure waves that develop during sudden releases of energy with a specific time-to-pressure curve profile ,19. The Micro RNAs (miRNA) are a type of ribonucleic acid approximately 22 nucleotides long. miRNAs modulate gene expression at the mRNA level, by either promoting the degradation of the target mRNA or by inhibiting translation. Each miRNA can have multiple regulatory targets . FurtherMechanical stimulation of cells via shockwaves has recently been reported to increase exosome release . These nAfter ethical approval of the experimental protocol (TVA-Nr.: MA58/005956/2012/6) by the Animal Protocol Review Board of the City Government of Vienna, male Sprague-Dawley rats weighing 200–250 g were obtained from Janviers Labs . The animals were provided with ad libitum access to food and water and were housed under simulated daylight conditions with alternating 12-h light–dark cycles. Rats were familiarized with the new environment for a period of one week. One day before the operation, every animal was allocated randomly to one of the treatment or control groups , and pre-operative BBB  baseline data were obtained. We defined exclusion criteria for this study based on the BBB-Score to identify animals that did not have a sufficient (BBB on day 3 > 7 or at week 1 > 13) or too strong (BBB < 10 at week 2) contusion to the spinal cord. This led to the exclusion of n = 6 control animals, n = 8 from the subacute group and n = 4 from the chronic treatment group.In this study, a spinal cord contusion injury model using the Infinite Horizon (IH) impactor  was utilized. The rats were first exposed to a 3% isoflurane gas/air mix, which induces a short general anesthesia. They were then injected intraperitoneally with Ketasol  and Rompun , according to their bodyweight, for maintaining anesthesia. Anesthetic depth was monitored through the toe pinch test, oxygen saturation, heart rate and breathing rate. If needed, additional low-dose isoflurane gas/air mix (0.5–1%) was used for deepening anesthesia. By using a rectal probe, core body temperature was measured and maintained at 37 °C through the use of an adjustable heating pad during the surgery.Temperature support was continued until recovery from anesthesia. After wound incision, the lamina arcus vertebrae of the 11th thoracic vertebra were prepared and a laminectomy was performed by drilling a small hole, slightly larger than the 2.5 mm tip of the IH impactor, into the lamina. That hole was then slightly enlarged and cleared of bone debris using a small bone rongeur. Afterward, the animal’s spinal column was held in place by clamping it rostrally and caudally to Th11 with the impactor’s stabilizing forceps. Using the hand wheels at the front and the side, the steel rod was positioned exactly 3–5 mm cranial to the laminectomy hole. Finally, all animals were subjected to an impact with a defined force of 150 kdyn. Wound closure was performed in anatomical layers using absorbable 4/0 sutures.All surgical procedures were performed under sterile conditions. Post-operative care was provided to all animals equally via subcutaneous injections of 10 mL of saline, analgesics and two antibiotic treatments . This treatment was given for the first 3 days after surgery, and the rat’s bladder was manually expressed until spontaneous urination returned.2 and a frequency of 5 Hz (Focused shockwave applicator: OE50) onto the area of surgery at the 11th thoracic vertebra. Based on available literature ,34,35, aFor evaluation of hind limb motor function recovery and coordination between fore- and hindlimbs after SCI and ESWT, we used an open field walking test, the BBB-Score, which is a well-established locomotor rating scale used in the field of spinal cord research. In short: rats were placed on an open field  and each hindlimb was assessed separately using a scale from 0 to 21 while the rat moved freely for at least 4 min. The test was conducted by two independent blinded observers before injury on day 3 post-injury and once per week throughout the observation period. The mean values of left and right hind limb scores were used to analyze changes before and after treatment . After aIn order to evaluate the three-dimensional appearance of spinal cords after the respective observation periods, spinal cords bearing contusion injury were extracted with the bone of the spinal column intact. The spinal column was stained in 50 mL centrifuge tubes filled with 1% Lugol’s Iodine for 48 h at 4 °C on a roller mixer. If the staining duration was determined to be insufficient , the staining was continued in 24 h increments, replacing the staining solution each time, until the spinal cord was fully stained. Scans were performed in a SCANCO µCT 50  in the residual staining solution at 90 kVp with 200 µA with a 0.5 mm Al Filter. The 1000 Projections/180° were integrated for 500 ms with a FOV of 35.2 mm. The scans were reconstructed to an isotropic resolution of 17.2 µm. A height of approximately 27 mm was scanned with the defect in the center of the scan . SubsequAfter µCT scanning, histological and immunohistochemical analyses were performed. Lugol’s iodine solution was washed out using 70% ethanol. Samples were rinsed in water for one hour and then decalcified in 10% EDTA for several weeks. The correct position for the histological sections was obtained with the help of µCT images. Embedding in paraffin was performed via the intermedium xylene after dehydration of the samples in an ascending ethanol series, beginning with 50% ethanol. Samples were cut into 4 µm thin sections and dried in a 37 °C incubator overnight. Standard H&E staining was performed as well as staining for Neurofilament (NF) and Luxol Fast Blue stainings.Luxol Fast Blue staining was performed with a Luxol staining solution in 96% EtOH . After that, the sections were differentiated with lithium carbonate, which results in a crisp myelin staining in blue. Then, the Nissl bodies were stained with cresyl violet .For NF staining, slides were steamed for 20 min in sodium citrate buffer (pH = 6) for 20 min. Afterward, sections were incubated with Bloxall (VectorLabs) for 10 min. The incubation with primary antibodies  was conducted in a humified chamber at room temperature for 1 h. Secondary antibodies, anti-mouse (BrightVision poly HRP), were incubated for 30 min, and the detection of the signal was obtained using ImmPACT Nova Red (VectorLabs). Finally, all sections were counterstained using Mayer’s Hematoxylin and permanently embedded.For analysis, slides were scanned with the Vectra Polaris Automated Quantitative Pathology Imaging System  and evaluated with HALO v3.4.2986.170 software . The Multiplex IHC v3.0 module was used for NF staining assessment. Representative areas were marked to generate a random forest classifier for analysis of Luxol stained slides.p-value and effect size (log2 fold change). The new miRNA-arrays consisted of 21 promising candidates (each treatment group had a slightly different array with 11 common miRNAs), which were analyzed at 5 different timepoints: preinjury, pre-treatment (week 2 for the subacute group and week 5 for the chronic), 1 h after the first treatment, 1 h after the third treatment before sacrificing the animals. Blood sampling for the controls was performed at the corresponding timepoints.In order to evaluate changes in the miRNA expression profile in blood serum of shockwave-treated animals, we screened for differential expression of 187 miRNAs in 8 animals . We used samples of the same animal before and 1 h after ESWT in both treatment groups. These miRNAs were determined by researching keywords such as inflammation/anti-inflammatory, angiogenesis, neuronal development/expression/enrichment/protection, motor function, and overall good detection in serum. Promising candidates for further validation in our experimental setup were chosen according to the Total RNA was extracted from 100 µL serum using the miRNeasy Mini Kit . Samples were thawed on ice and centrifuged at 12,000× g for 5 min to remove any cellular debris. For each sample, 200 µL of serum was mixed with 1000 µL Qiazol and 1 µL of a mix of 3 synthetic spike-in controls . After a 10-min incubation at room temperature, 200 µL of chloroform was added to the lysates, followed by cooled centrifugation at 12,000× g for 15 min at 4 °C. Precisely 650 µL of the upper aqueous phase was mixed with 7 µL glycogen (50 mg/mL) to enhance precipitation. Samples were transferred to a miRNeasy mini-column where RNA was precipitated with 750 µL ethanol followed by automated washing with RPE and RWT buffer in a QiaCube liquid handling robot. Finally, total RNA was eluted in 30 µL of nuclease-free water and stored at −80 °C to await further analysis.Starting from total RNA samples, cDNA was synthesized using the miRCURY LNA RT kit . Reaction conditions were set in accordance with the manufacturer’s specifications. In total, 2 µL of total RNA was used per 10 µL reverse transcription (RT) reaction. To monitor RT efficiency and presence of impurities with inhibitory activity, a synthetic RNA spike-in (cel-miR-39-3p) was added to the RT reaction. PCR amplification was performed in a 384-well plate format in a Roche LC480 II instrument  using a miRCURY LNA SYBR Green PCR kit  with the following settings: 95 °C for 10 min, 45 cycles of 95 °C for 10 s and 60 °C for 60 s, followed by melting curve analysis. To calculate the cycle of quantification values , the second derivative method was used. Spike-in control values were used for monitoring data quality. Serum samples with low RNA recovery assessed by spike-in levels were not included in the analysis. Data normalization was performed using the RNA spike-in controls by subtracting the individual miRNA Cq-value from the RNA Spike-In Cq-value, thus obtaining delta-Cq (dCq) values that were used for the analysis. Hemolysis was assessed in all samples using the ratio of miR-23a-3p versus miR-451a, and a cut-off of dCq > 7 was applied to indicate a high risk of hemolysis .Missing values were imputed with 42, representing the lowest detectable amount of miRNA in the sample, before normalization. After imputation, outliers were identified via Grubb’s test at a significance level of 5 % and excluded. Furthermore, serum samples with a Uni-Spike-In 4 Cq value more than 3 standard deviations above the arithmetic mean of all the other samples were excluded due to low RNA recovery. This led to the exclusion of the pre-surgery sample of individual 91 (control group), which was then imputed with the arithmetic mean of the Cq values of the other individuals in the same experimental group, setting, timepoint and miRNA because otherwise only two values would remain in that experimental group, and because the pre-surgery Cq-values can be reasonably assumed to be similar.In order to identify the miRNAs with the greatest fluctuations and therefore to gather potential insights into the most important ones in regard to SCI and ESWT, we performed PCA on the miRNA RT qPCR across groups and timepoints. After removing all rows (sample-timepoint combinations) with missing values, a PCA of baseline normalized miRNA ∆Cq values was performed via the prcomp function from the stats package in R  with cenBased on the first few principal components cumulatively describing at least 80% of total variance, the relative feature importance of each miRNA was calculated as follows:In the interest of finding miRNAs most closely related to SCI, we performed hypothesis tests on the difference between miRNA levels—as quantified via RT qPCR—at the pre- and post-surgery timepoints. These diagnostic hypothesis tests were performed using the wilcox.test function from the stats package in R . These tA repeated measures two-way ANOVA was performed to assess the treatment effect and differences between ESWT and control groups at individual observational timepoints. Geisser Greenhouse and Šidák corrections were applied to correct sphericity and multiple comparisons. In the case of missing values, a mixed-effects model was automatically applied using Graphpad.Spearman’s correlation coefficient between BBB-Scores and miRNA serum levels at the respective weeks was calculated with Graphpad Prism 9.2.0. Analysis was performed separately for the ESWT treated and control group, as well as for the data from both groups combined. Additionally, for miRNAs analyzed in subacute and chronic treatment settings, pooled data were used for assessment.In this study, we evaluated the effects of ESWT on functional regeneration in subacute and chronic SCI. Throughout the observation period after injury and treatment, continuous assessments of changes in motor function were conducted to observe the effects of ESWT on functional regeneration. We found that immediately after treatment with ESWT, rats of both treatment groups experienced an improvement in coordination, better paw positioning and toe clearance, whereas control animals, except for one outlier, reached a plateau of BBB = 12–13 and did not improve further . AnimalsAt the end of the observation period, we aimed to analyze structural and dimensional parameters. In order to account for the spatial complexity of secondary spinal cord injuries, we implemented three-dimensional imaging of the contused spinal cord using ex-vivo µCT. μCT is widely used for the study of calcified tissue, but a similar use for soft tissues is hindered by their low X-ray attenuation. This limitation was overcome in this study by the use of Lugol’s solution as contrast agents . OutcomeIn In order to complement 3D µCT-derived morphological findings, histology and immunohistochemistry of spinal cord tissue were conducted and compared. Analysis revealed significant loss of axons as well as demyelination in the affected area of the spinal cord following contusion. A general reduction in spinal cord diameter was noticeable, as already observed in the μCT evaluation. Trained algorithms for the automatic detection of NF-positive axons as well as Luxol-positive myelin performed with a high accuracy and enabled quantitative analysis of the histological slides . CompariContrary to observed functional improvement and previously published data, we did not observe any significant morphological differences, e.g., spared white matter, after ESWT in either treatment group compared to controls. This finding was consistent with the aforementioned automatically measured spinal cord parameters in µCT imaging data, indicating an underlying effector mechanism of ESWT in addition to neuroprotective or regenerative mechanisms.p-value (<0.05) and effect size  and effect size (log2 fold change) before and after ESWT for both treatment setups for further validation. Out of these, 11 miRNAs were common to both arrays.In order to elucidate underlying mechanisms after SCI and ESWT, we performed an analysis of systemic microRNAs and their potential contributions to regeneration at defined timepoints of the observation period. The initial screening for ESWT-associated changes in miRNA profiles  includedIn the subacute experimental setting, seven potentially therapeutically relevant miRNAs were identified. Differences between the ESWT treatment and control group for pooled timepoints (column factor in two-way ANOVA) were observed for hsa-miR-100-5p and hsa-miR-136-5p. Furthermore, rno-miR-148b-5p, rno-miR-214-3p, hsa-miR-28-5p, hsa-miR-218-5p, hsa-miR-199a-3p and hsa-miR-375 showed differences between groups at repeated measures corrected weekly compared to after correction for repeated measures. Diagnostic markers, in terms of statistically significant differences between baseline values and miRNA expression levels at week two, were observed for hsa-miR-199a-3p, hsa-miR-375 and hsa-miR-152-3p.In the chronic experimental setting, four miRNAs were potentially therapeutically relevant. These include hsa-miR-100-5p, hsa-miR-124-3p, hsa-miR-218-5p, with a significant difference between groups at the endpoint and hsa-miR-124-3p as well as hsa-miR-382-5p with significant differences between pooled timepoints. Additionally, nine miRNAs were identified as possible diagnostic markers, including hsa-miR-218-5p, hsa-miR-125-5p, hsa-miR-133b, hsa-miR-144-3p, hsa-miR-152-3p, hsa-miR-199a-3p, hsa-miR-29c-3p, rno-miR-148-5p and rno-miR-381-3p.p-values as well as the mean and standard deviation over time are reported in Corresponding p = 0.0003) in the subacute ESWT group for hsa-miR-375  and in the control group for hsa-miR-146a-5p  and hsa-miR-29c-3p . In the chronic treatment set up, no correlations with r > 0.5 were observed.In order to investigate the influence of the examined microRNAs on motor function, Spearman’s correlation coefficients were calculated. Correlation coefficients r > 0.5 were observed in the pooled data from subacute and chronic control groups (ESWT and control combined) for hsa-miR-29c-3p   altered Another identified miRNA is miR-214-3p. Controversy exists in the literature about the role of miR-214-3p in neuroprotection. Yuanzhi Fan et al. showed neuroprotective effects after the application of tetramethylpyrazine in connection with the downregulation of miR-214-3p in SCI after 21 days . In accoIn the subacute treatment setting, miR-218-5p was significantly upregulated at the end point, which is in agreement with decreased dopaminergic neuron loss in Parkinson’s disease patients due to miR-218-5p targeting LASP1 . In contSignificant downregulation of miR-199a-3p after SCI constitutes this miRNA as a potential marker for SCI, as also observed by other authors ,59,60. FIn response to SCI, miR-375 was downregulated at week two, making it a potential candidate for a diagnostic marker after SCI. Furthermore, directly after the first ESWT application, as well as at all successive timepoints, miR-375 was upregulated in the ESWT group. Various pathways have been described for motor neuron development in the spinal cord in response to miR-375 expression, such as targeting the CCND2 and PAX6. Additionally, neuroprotective effects of miR-375 through p53 inhibition have been described in patients suffering from spinal muscle atrophy . In anotAnother potential diagnostic marker, miR-152-3p, which was significantly downregulated after SCI in our study, has already been documented by Ding et al. .In the chronic experimental setting, we identified eight miRNAs , which were significantly downregulated after SCI, and one (miR-133b) which was upregulated. After treatment with shockwaves, four miRNAs  were significantly altered in their expression profile.According to the literature, miR-100-5p has previously been described to be involved in the pathogenesis of neurodegenerative diseases ,71,72,73Levels of circulating miR-124-3p were significantly lower in the ESWT group compared to the control group. miR124-3p has widely been researched for its involvement in several pathologies of the CNS ,85,86,87One of the most promising miRNAs in our study was the miR-382-5p. While the control animals demonstrated unaltered expression levels throughout the entire experiment, animals in the ESWT group experienced a significant downregulation by the time of the third shockwave treatment, with this effect being even more pronounced at the end of observation time. In a study by Xiang et al., an abnormal downregulation of lncRNA Ftx and Nrg1 and upregulation of miR-382-5p after SCI was observed, which contributed to the inflammatory response in microglial cells and affected SCI repair . PhosphaIn our chronic experimental setting, miR-218-5p was found to be significantly downregulated at the end of the observation time . MmiR-21These findings, in conjunction with our own data, could provide insight into possible underlying mechanisms leading to observed functional recovery after SCI and ESWT treatment at subacute and chronic timepoints post-injury.However, limitations of this study have to be noted. Our explorative study did not include a sham operated group. Furthermore, functional improvement within the chronic setting was still ongoing by the end of the observation period, which led to the suggestion of a prolonged phase of observation in future studies. Furthermore, further investigations are required to explore the effects of the laminectomy on miRNA changes, as improved bone regeneration due to ESWT could also contribute to some of the observed changes in miRNA levels. Substantial financial funding is required to advance miRNA analysis with a larger number of implications. Due to limited miRNA recovery in some samples during qPCR, some values needed to be excluded from the analysis.This study demonstrates the functional improvement of ESWT-treated animals after SCI in a subacute and chronic treatment setting. Our results suggest that this functional improvement was not based on morphological changes. However, significant changes in circulatory levels of miR-382-5p and miR-375 upon ESWT were observed. This could provide further insights into underlying mechanisms regarding their influence on neuroplasticity as well as inflammatory response, hence potentially providing targets for therapeutic intervention in future studies. Results of this study were fundamental for a recently started two-arm three-stage adaptive, prospective, multi-center, randomized, blinded, placebo-controlled clinical trial on the effects of ESWT in acute traumatic spinal cord injury .